Method of breeding glyphosate resistant plants

ABSTRACT

A method of producing a plant which shows resistance to a herbicide in particular glyphosate, said method comprising  
     (i) applying said herbicide to a population of herbicide resistant plants at an advanced vegetative stage before flowering,  
     (ii) using pollen from said plants to fertilise female plants; and  
     (iii) obtaining progeny therefrom.  
     This method can be used in various ways, including in the production of pure hybrids, as a “switching” system in particular for sterility/fertility in plants and in the production of transgenic plants generally.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This is a divisional of U.S. application Ser. No. 09/100,516 nowpatented U.S. Pat. No. ______, filed on Jun. 19. 1998.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of producing herbicideresistant plants. This method is useful in the production of hybrids aswell as in the production of transgenic plants generally.

BACKGROUND OF THE INVENTION

[0003] Incorporation of a gene which confers resistance to theherbicides such as glyphosate into plants is well known (see for exampleGasser et al., Recent Adv Phytochem, New York, Plenum Press 1988 22,p45-59) Such plants, in particular corn plants, are available on themarket, for example from DeKalb Genetics. In the case of glyphosateresistance, the plants include a gene which brings about aglyphosate-resistant event, GA21, which is based on the gene encoding analtered an altered active site enzyme.

[0004] For example diplid plants may be homozygous (termed RR where R isglyphosate resistance) or heterozygous Rr for glyphosate resistance,depending upon their method of production and lineage.

[0005] When used in plant breeding programmes, heterozygous plants willresult in the production of a significant proportion of plants which donot possess the herbicide resistance trait. For example, when dealingwith glyphosate resistant plants, this may be representeddiagrammatically by:

Rr*Rr>RR+2 Rr+rr

[0006] R=the transgene and r-azygous wildtype

[0007] Where one of the parents does not carry the resistance trait,even more of the progeny will lack the trait i.e.

rr*Rr>rR+rr

[0008] This means that heterozygous plants cannot normally be used asgene donors in the production of commercial seed for farmers to grow,because much of the seed would lack the trait, and desired levels ofpurity would not be achieved.

[0009] However, the use of heterozygous plants could actually bepreferred to the use of homozygotes in seed production. This is becausethe phenomenum of gene silencing, in which transformation events becomeunstable or stop functioning after several generations, occurs morefrequently in homozygous plants.

[0010] There is a need for a process whereby the yield of plantsdemonstrating a desired trait, such as herbicide resistance, isincreased from breeding programmes which include heterozygous plants.

SUMMARY OF THE INVENTION

[0011] The applicants have found that by applying a herbicide at aparticular stage of plant development, maintenance of the herbicideresistance in the pollen and therefore in the progeny can be enhanced.

[0012] The invention provides a method of producing a plant which showsresistance to a herbicide, said method comprising

[0013] (i) applying said herbicide to a population of herbicideresistant plants at an advanced vegetative stage before flowering,

[0014] (ii) using pollen from said plants to fertilise female plants;and

[0015] (iii) obtaining progeny therefrom.

[0016] The method of the invention may be applied to plants which areresistant to various herbicides. In particular it may be applied tonon-selective herbicides in which the method of plant resistance to theherbicide is achieved through a modified endogenous gene which producesa molecule that is unaffected by the presence of the herbicide.

[0017] A particular herbicide for use in the method of the invention isglyphosate and salts thereof such as those sold as Roundup™ andTouchdown™. Other herbicides which may be used in this way areAcetolactate Synthase (ALS) and (AHAS) inhibitors (sold as Pursuit™ andSceptre™), where the herbicide resistant mechanism of the plant isalteration of the active site as described above.

[0018] The invention is particularly applicable where the plants arecrop plants such as corn.

[0019] What constitutes a suitable “advanced vegetative stage” may varyfrom plant to plant, depending upon the pollen development. This may bedetermined in each case using routine test methods. However in general,it will comprise a stage equivalent to V5 or above, for example up toV16 in corn. A description of these growth stages in corn is given by S.W. Ritchie et al., “How a Corn Plant Develops. Rev. Feb. 1982” SpecialReport (Iowa State University of Science and Technology, CooperativeExtension Service, No. 48) 1982, 21 p: col.ill;, the content of which isincorporated herein by reference.

[0020] The invention is based upon the statistically significantdeviance of observed segregation from expected segregation that observedin series of experiments. The glyphosate resistance allelic, designatedas R, behaves in a dominant fashion. Therefore, plants either homozygousR (i.e. RR) or heterozygous R (i.e. Rr) will be resistant to foliarapplications of glyphosate. Starting from a known allelic composition,the observed segregation can be statistically compared with the expectedsegregation by using a Chi-Square test, a readily accepted statisticaltool for comparing the deviation of observed from expected values. Itwas found that, using this method of analysis, the segregation varied ina statistically significant manner.

[0021] Specifially, it has been observed that a corn plant which isheterozygous for the Glyphosate-resistance event GA21 behaves as if itwere RR when used as a pollen parent in a cross to another corn plantand when it has first been sprayed with glyphosate at a relativelyadvanced vegetative stage, but before flowering. Conversely, if the Rrplant is not sprayed with glyphosate, or if the spray is delivered at anearly vegetative growth stage, the plant behaves normally, andcontributes both R and r pollen to the cross.

[0022] Diagramatically:

[0023] With glyphosate rr*Rr>Rr only

[0024] Without glyphosate, or glyphosate early rr*Rr>rR+rr

[0025] Similar effects are not seen with female gametes, or seed set.

[0026] Without being limited by any theory or mode of actions, it isthought that residual herbicide within the plant kills, or inactivates,r pollen during development. This is consistent with the known mode ofaction of the herbicide glyphosate, which is translocated readily in theplant and which will accumulate at sites of high metabolic activity(sinks) such as the developing tassel and anthers. It is noteworthy alsothat GA21 is an alternative-enzyme method of protection, not a herbicidedegradation system, so GA21 itself should not affect the distributionand persistence of the herbicide in the plant.

[0027] The method of the invention can be used in several ways. In oneway, it may be used to improve the purity of inbred and hybrid seedproduction (especially herbicides resistant crops, by preventing theproduction of pollen lacking the desired genes.

[0028] Normal standards for seed production allow 2-4% offtypes inhybrid seed. However, if the crop contains a herbicide resistance trait,and the trait is not present in the offtypes, these will be killed whenthe farmer uses the herbicide for weed control. Death of these plantscan cause farmer concern and, theoretically at least, could somewhatreduce yield (to the extent that the offtypes, had they survived, wouldhave set grain). This has become a significant issue in the USA duringthe last couple of years.

[0029] One possible source of offtypes occurs when the male parent isused as the trait donor in a production field, and (using glyphosate asan example) contains either rr or Rr genotypes as well as the desiredhomozygote, RR. Obviously, we can use glyphosate itself to eliminate rrfrom the male rows, so they are not a problem. But, because the trait isdominant, the herbicide will not eliminate the Rr plants. However, usinga late vegetative spray of pollen as described above will eliminate ther pollen from these heterozygotes, so they will behave as RR. Hence, onesource of susceptible offtypes will be prevented.

[0030] Thus in a further aspect, the invention provides, in theproduction of herbicide resistant hybrid seed, a method of reducing thenumbers of herbicide susceptible offtypes in a population of herbicideresistant hybrids, said method comprising spraying male parent plantswith said herbicide at at late vegetative stage of growth such that anyheterozygotes amongst the population will produce pollen which showherbicide resistance as the dominant trait.

[0031] The method of the invention may also be used in the production oftransgenic plants. Frequently, herbicide resistance is used as aselectable marker to allow selection of successful transformants whichinclude a desired trait gene. In broad terms, plants are cotransformedwith the desired trait gene as well as a herbicide resistance gene. Thetrait gene and the herbicide resistance gene are preferably linked sothat both share similar fate in the transformation process. Applicationof herbicides to the transformants will eliminate unsuccessfultransformants which are not resistant to the herbicide, leaving onlythose plants which include the desired trait gene.

[0032] Using the method of the invention, linkage, and preferably tightlinkage, of the desired trait gene to the herbicide resistance gene willmaximise the production of trait containing pollen from male plant rows,even if the rows contain heterozygotes. The advantages associated withthe use of this pollen in breeding programmes are clear.

[0033] Thus in a further aspect the invention provides a method oftransforming a plant with a desired transgene, said method comprising

[0034] (i) transforming plant cells with:

[0035] (a) a construct which comprises a desired transgene;

[0036] (b) a herbicide resistance gene;

[0037] (ii) growing plants from said cells;

[0038] (iii) applying herbicide to plants grown in step (b) at anadvanced vegetative stage so as to ensure that the pollen produced fromsurviving plants carries herbicide resistance and the desired trait in adominant form;

[0039] (iv) using pollen produced in step (iii) to fertilise a femaleparent, and

[0040] (v) obtaining the progeny thereof.

[0041] Suitably the desired transgene and the herbicide resistance genewill be on the same construct prior to the transformation, althoughco-transformation procedures as are known in the art, may also be used.

[0042] This method is particularly advantageous in that it delivers adesired trait at high purity from the male parent.

[0043] Desired transgenes are well known in the art. They include geneswhich control sterility and/or fertility as well as genes which causequality traits such as high oil.

[0044] This effect can be used in hybridization technology inparticular. For example, by using herbicide resistance such asglyphosate resistance as a selectable marker/part of the construct for amale gametophyte killer gene (male sterility gene S), plants ofstructure RSrs can be created. If this is sprayed with herbicide at anadvanced vegetative stage as described herein, it will be totally malesterile. This is because the S gene sterilises the R pollen, and theherbicide, such as glyphosate itself disables the r pollen. Thus, thiscan be used as a sterile female parent in a production field, thuseliminating the need for expensive hand-detasseling

[0045] Conversely, if the plant is grown in the absence of herbicide, itwill still produce r pollen. Thus the supplies of the heterozygote canbe increased by growing it in a normal isolated increase field withoutherbicide, such as glyphosate, in which case it will set a 1:1 ratio ofRSrs and rsrs seed.

[0046] The unwanted rsrs types which will also be produced in thisprocess can be readily removed in the next generation, for example byapplication of the herbicide

[0047] In order to increase parent seed from one generation to the next,we just spray glyphosate at an early growth stage which will eliminatethe susceptible plants, but will not affect fertility of the survivingheterozygotes.

[0048] If the plants are required as female parents in a productionfield, they would be sprayed both early and late, first to eliminatesusceptible plants, and second to enforce male sterility in thesurvivors. In such a system, the herbicide could be regarded as actingas a “switch” for male sterility.

[0049] Hence, in a further aspect, the invention provides a method ofproducing plants which are reversibly male sterile, said methodcomprising

[0050] (i) transforming a plant with a construct which comprises a malegametophyte killer gene (S) and a herbicide resistance gene (R),

[0051] (ii) selecting a transformant which is heterozygous with respectto said genes of structure RSrs, and either

[0052] (iii) either (a) where male sterile plants are required, applyingherbicide to plants grown from said transformants at an advancedvegetative stage so as to disable residual fertile pollen therein, or

[0053] (b) where male fertile plants are required, growing said plantsin the absence of herbicide at the advanced growth stage.

[0054] Selection at stage (ii) is suitably carried out by applyingherbicide at an early growth stage, as outlined above.

[0055] In this system, it would be helpful if the male parent in theproduction field also carried similar herbicide resistance, to avoidproblems from spray drift onto a susceptible parent line.

[0056] This process allows the maintainance/increase of parent seed in asimple increase field rather than as a crossing block, which is requiredfor traditional male sterility systems e.g. CMS (cytoplasmic malesterility).

[0057] For some crops such as wheat, it is highly desirable to be ableto interplant the male with the female, rather than have the parents inseparate rows (movement of pollen from one plant to another is a problemfor hybrid wheat). In order to achieve this, the female parent linesuitably carries an additional, different dominant, herbicide resistance(e.g. Liberty), whilst the male only carries only one such as glyphosateresistance. Both early and late sprays of the common herbicide inparticular glyphosate would be carried out as before in order to producemale sterile females and to produce pollen which included dominantglyphosate resistance. Subsequently application of the additionalherbicide, such as Liberty, post flowering would kill off the maleplants.

[0058] In one particular embodiment of the invention, traits like highoil, which are expressed in the grain, could be partially switched byglyphosate. For this purpose, glyphosate resistance is used as aselectable marker/part of the construct/tightly linked to the qualitytrait, and the quality trait is delivered by the pollinator (which it isin the top-cross high oil system). Then, a heterozygous pollinator wouldeither deliver pure trait pollen, or a 1:1 mixture of trait andnon-trait pollen, depending on whether it was sprayed with glyphosate atthe appropriate time.

[0059] A further application of the method of the invention is in theavoidance of gene silencing for transgenes.

[0060] A major problem for transformation technology is that manytransformation events become unstable, have adverse side effects on thehost plant, or stop functioning, several generations down the track fromthe primary transformant plant. This necessitates production of manyevents and empirical screening to find ones which are stable anduseable: it is time consuming, costly, and, at present, probably therate-limiting step to the technology.

[0061] It has been observed that problems most frequently arise whenplants are created which are homozygous for the introduced trait (TT).Normally, it is necessary to have such plants in order to create aparent which will deliver the trait in hybrid production.

[0062] Using the method of the invention, the production of homozygousplants may be avoided. Using the herbicide resistance such as glyphosateresistance as a selectable marker, it is necessary only to produce andmaintain a heterozygous parental line RTrt (provided this line is usedas the male in a hybrid cross). As noted above, use of herbicide such asglyphosate, sprayed selectively on the male parent in the productionfield, will make it function as the homozygote RTRT, although theproblems associated with normal homozygotes in terms of gene silencingare avoided.

[0063] Thus in yet a further aspect, the invention provides a method ofproducing a transgenic plant which demonstrates a desired trait, whichmethod comprises crossing to a female plant which lacks the desiredtrait, with pollen obtained from a male plant which is heterozygous forsaid desired trait and also for a herbicide resistance, and applyingherbicide to the progeny at an advanced stage of vegetative growth inorder to ensure that the trait and herbicide resistance are dominant inthe pollen.

[0064] In this way, the progeny from the cross will segregate Rr or rr.The rr can be removed in the next generation by application ofherbicide, thus leaving just the Rr plants. After treating withherbicide in a manner that selectively kills the r gametes, those plantscan then be used a males in another cross to the female lacking thetrait, producing the next cycle of Rr and rr progeny.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The following examples illustrate the invention.

EXAMPLE 1

[0066] Confirmation of Heterozygosity and Establishment of GlyphosateTreatment Classes

[0067] Progeny of a cross between a homozygous resistant plant (RR) anda homozygous susceptible plant (rr) were planted at the Garst ResearchCentre near Slater, Iowa, and at the Hawaii Research Centre near Kunia,Hi. It was expected that none of the progeny would segregate forresistance to foliar applications of glyphosate. To test this, theprogeny growing at the IA test site were sprayed with glyphosate at arate of 64 oz per acre during the V5 and V6 stages of growth (26 daysafter planting). At the same time and rate, several plants in a row ofknown susceptible plants were sprayed. All of the sprayed plots wereexamined for plant mortality 7 days after spraying. As expected, all ofthe susceptible check plants were dead and while all of the putativeheterozygous resistant plants were alive (Table 1). TABLE 1 Observed andexpected frequencies among heterozygous resistant plants treated withglyphosate during Experiment 1. Expected Observed Class SegregationSegregation Heterozygous Resistant All Resistant All Resistant NoneSusceptible Homozygous Susceptible All Susceptible All Susceptible NoneResistant

[0068] A second application of 32oz per acre was applied to theheterozyous resistant plants at the V7 to V8 stage of growth (9 dayslater). These plants comprised the “Sprayed” class in subsequentexperiments. In HI, none of the plants were sprayed with glyphosate andthese plants comprised the “Unsprayed” class in subsequent experiments.At both locations, crosses were made between the heterozygous resistantplants and homozygous susceptible plants.

Example 2

[0069] Evaluation of Progeny from Sprayed and Unsprayed Classes

[0070] Progeny of the crosses from each class were planted at the HIlocation. All progeny plants from each class were sprayed withglyphosate at a rate of 64 oz per acre during the V3 to V4 stages ofgrowth. Ten days post-application, counts of resistant and susceptibleplants were made for each class. The expected segregation, 1:1 resistantto susceptible, was compared to the observed segregation for each class(Table 2). Segregation among the progeny from the Unsprayed class didnot differ significantly from the expected segregation (Chi-square=2.12,d.f.=1, Prob>0.1). However, segregation among the progeny from theSprayed class differed significantly from the expected segregation(Chi-square=669.34, d.f.=1, Prob<0.01). TABLE 2 Observed and expectedfrequencies among Sprayed and Unsprayed classes during Example 2. ClassExpected Observed (Total Plant Evaluated) Segregation SegregationSprayed (504) 252 Resistant: 504 Resistant:0 252 SusceptibleSusceptible** Unsprayed (182) 91 Resistant: 91 100 Resistant:82Susceptible Susceptible

[0071] The absence of susceptible progeny among the Sprayed classprovides clear evidence that treating heterozygous resistant plants withhigh doses of glyphosate at V5 and later plant growth stages imposes aselection that favours the exclusive production of resistant pollen.

1. A method of producing a plant which shows resistance to a herbicide,said method comprising (i) applying said herbicide to a population ofherbicide resistant plants at an advanced vegetative stage beforeflowering, (ii) using pollen from said plants to fertilise femaleplants; and (iii) obtaining progeny therefrom.
 2. A method according toclaim 1 wherein the herbicide resistant plants are glyphosate resistant,and the herbicide applied in stage (i) is glyphosate.
 3. A methodaccording to claim 1 wherein the plants comprise crop plants.
 4. Amethod according to claim 3 wherein the crop plants comprise corn.
 5. Amethod according to claim 4 wherein in step (i), the herbicide isapplied at the V5 stage of growth or later.
 6. A method according toclaim 1 wherein the progeny comprise herbicide resistant hybrid seed. 7.A method according to claim 1 wherein the plants contain a furtherdesired transgene.
 8. A method according to claim 7 wherein the furthertransgene is a gene which encodes a quality trait which is deliverableby a pollinator.
 9. A method according to claim 8 wherein the qualitytrait comprises a high oil system.
 10. A method according to claim 7wherein the transgene is a fertility/sterility controlling gene.
 11. Amethod according to claim 10 wherein said fertility/sterilitycontrolling gene is a male sterility gene.
 12. In the production ofherbicide resistant hybrid seed, a method of reducing the numbers ofherbicide susceptible offtypes in a population of herbicide resistanthybrids, said method comprising spraying male parent plants with saidherbicide at an advanced vegetative stage of growth such that anyheterozygotes amongst the population will produce pollen which showherbicide resistance as the dominant trait.
 13. Hybrid seed obtained bythe method of claim
 12. 14. A method of producing plants which arereversibly male sterile, said method comprising (i) transforming a plantwith a construct which comprises a male gametophyte killer gene (S) anda herbicide resistance gene (R), (ii) selecting a transformant which isheterozygous with respect to said genes of structure RSrs, and either(iii) either (a) where male sterile plants are required, applyingherbicide to plants grown from said transformants at an advancedvegetative stage so as to disable residual fertile pollen therein, or(b) where male fertile plants are required, growing said plants in theabsence of herbicide at the advanced growth stage.
 15. A methodaccording to claim 14 wherein the selection at stage (ii) is carried outby applying herbicide at an early growth stage.
 16. A transgenic plantor seed obtained by the method of claim
 14. 17. A method of transforminga plant with a desired transgene, said method comprising (i)transforming plant cells with: (a) a construct which comprises a desiredtransgene; (b) a herbicide resistance gene; (ii) growing plants fromsaid cells; (iii) applying herbicide to plants grown in step (b) at anadvanced vegetative stage so as to eliminate any non-transformed plantsand to ensure that the pollen produced from surviving plants carriesherbicide resistance and the desired trait in a dominant form; (iv)using pollen produced in step (iii) to fertilise a female parent, and(v) obtaining the progeny thereof.
 18. A plant or seed obtained by themethod of claim
 17. 19. A method of producing a transgenic plant whichdemonstrates a desired trait, which method comprises crossing to afemale plant which lacks the desired trait, with pollen obtained from amale plant which is heterozygous for said desired trait and also for aherbicide resistance, and applying herbicide to the progeny at anadvanced stage of vegetative growth in order to ensure that the traitand herbicide resistance are dominant in the pollen.
 20. A plant or seedwhich is produced by a method according to claim 19.